To examine the role of Ca2+ in early neuronal death, we studied the impact of free intracellular calcium concentration ([Ca2+]i) on survivability in populations of cultured mouse spinal neurons. We asked whether early neurotoxicity was triggered by Ca2+ influx, whether elevated [Ca2+]i was a predictive indicator of impending neuronal death, and whether factors other than [Ca2+]i increases influenced Ca2+ neurotoxicity. We found that when neurons were lethally challenged with excitatory amino acids or high K+, they experienced a biphasic [Ca2+]i increase characterized by a primary [Ca2+]i transient that decayed within minutes, followed by a secondary, sustained, and irreversible [Ca2+]i rise that indicated imminent cell death. We showed that in the case of glutamate-triggered neurotoxicity, processes triggering eventual cell death required Ca2+ influx, and that neurotoxicity was a function of the transmembrane Ca2+ gradient. Fura-2 Ca2+ imaging revealed a "ceiling" on measurable changes in [Ca2+]i that contributed to the difficulty in relating [Ca2+]i to neurotoxicity. We found, by evoking Ca2+ influx into neurons through different pathways, that the chief determinants of Ca2+ neurotoxicity were the Ca2+ source and the duration of the Ca2+ challenge. When Ca2+ source and challenge duration were taken into account, a statistically significant relationship between measured [Ca2+]i and cell death was uncovered, although the likelihood of neuronal death depended much more on Ca2+ source than on the magnitude of the measured [Ca2+]i increase. Thus, neurotoxicity evoked by glutamate far exceeded that evoked by membrane depolarization with high K+ when [Ca2+]i was made to increase equally in both groups. The neurotoxicity of glutamate was triggered primarily by Ca2+ influx through NMDA receptor channels, and exceeded that triggered by non-NMDA receptors and Ca2+ channels when [Ca2+]i was made to rise equally through these separate pathways. The greater neurotoxicity triggered by NMDA receptors was related to some attribute other than an ability to trigger greater [Ca2+]i increases as compared with other Ca2+ sources. We hypothesize that this represents a physical colocalization of NMDA receptors with Ca(2+)-dependent rate-limiting processes that trigger early neuronal degeneration.